This invention relates in general to a method for the verification of polymerase chain reaction (PCR) amplification, and relates more specifically to a separationless electrochemical method whereby the execution of PCR is rapidly verified.
A few hundred molecules of DNA can now be amplified by the polymerase chain reaction (PCR). Since the introduction of PCR, the time required for its execution has been considerably reduced and has now shrunk to minutes. Because a substantial fraction of the attempted PCRs do not provide the sought amplification, verification of the success of PCR is an essential process element. The practitioner of PCR must know before further using the sample prepared that the DNA was amplified as expected. Kopp, et al. (Science 1998, 280, 1046-7) recently carried out a rapid continuous-flow polymerase chain reaction (PCR) on a chip, amplifying a segment of the DNA of Neisseria gonorrhoeae. Using a micromachined chemical amplifier, they amplified, through 20 cycles, a 176-base DNA segment in periods ranging from 1.5 to 18.7 minutes. Such rapid amplification is relevant to on-site medical diagnostics and to prompt identification of environmental biological hazards, including biological warfare agents. Verification of the amplification, in a time period that is not longer than the amplification period itself, is of importance in the operation of the amplification-dependent analytical systems, because, in the absence of sufficiently rapid verification, the overall time required is limited by the duration of the verification process. Amplification of a particular DNA molecule has been usually confirmed in a two step assay, the first of which is the separation of the DNA (e.g. by gel electrophoresis) and the second of which is the detection of the separated DNA by a photonic method, based on intercalation of a light-absorbing dye or a fluorescent, chemiluminescent, or electrochemiluminescent compound and measuring the absorbed or emitted photon flux. The separations of the first step could not be carried out in minutes. One of the fastest separation methods, high speed capillary gel electrophoresis (Effenhauser, et al., Anal. Chem 1994, 66, 2949), still required a complex and time-consuming setup procedure.
A more rapid and simpler method for verifying amplification of DNA by PCR is needed. It is the objective of this invention to provide such a method.
The present invention provides a separationless electrochemical method whereby PCR amplification is verified. The method utilizes an electrode coated with a redox polymer film. The redox polymer film is preferably a redox hydrogel. A binding agent is immobilized in the redox polymer film, preferably through covalent bonding of the binding agent to the redox polymer. The DNA is labeled, while amplified, with two or more different ligands, the first of which binds strongly to the binding agent immobilized in the redox polymer film. When the sample in which the amplification is to be confirmed is contacted with the electrode, amplified DNA is immobilized on the electrode through linkage of the immobilized binding agent in the redox polymer film with the first ligand. The presence of the amplified DNA on the electrode is detected through exposure of the electrode to a detection marker. The detection marker is a molecule with two functional groups. One of the functional groups binds with the second ligand of the amplified DNA; the second functional groups of the detection marker produces an electrochemically detectable signal. The second functional group of the detection marker is generally a catalyst, usually an enzyme. In the preferred embodiment the catalyst of the detection marker is an oxidoreductase. When the second ligand of the immobilized amplified DNA in or on the redox polymer film binds the detection marker, the catalyst of the detection marker comes into contact with the redox polymer and becomes an electrocatalyst. As a result, when the electrode is poised at an appropriate potential, a detection compound, which in the preferred embodiment, is a substrate for the oxidoreductase, is catalytically electroreduced or electrooxidized. The electrooxidation or the electroreduction results in an increase in the current passing through the electrode. The electrode is preferably poised at a potential where little or no current flows in absence of either the redox polymer or the detection marker in or on the film on the electrode. The detection compound may be added to the solution to be tested for the amplified DNA from the PCR, or it can be internally generated in the film, by reacting stable and preferably non-toxic reactants in catalytic centers incorporated in the film. The preferred detection compound generating catalytic centers in the film are enzymes, the presence of which does not substantially increase or decrease the current resulting from the electrooxidation or electroreduction of the detection compound.
In an example of such a method, a biotin labeled primer of one nucleotide sequence and a digoxigenin labeled primer of a complementary sequence are added to the DNA amplification solution. A redox polymer film, comprising avidin, is immobilized by crosslinking on a carbon electrode. Being biotin labeled, the amplified DNA binds to the avidin on the electrode. The presence of the amplified DNA is detected by exposing the electrode to a solution of horseradish peroxidase (HRP) labeled antidigoxin. Binding of the horseradish peroxidase labeled antidigoxin to the digoxigenin function of the amplified DNA converts the film on the electrode to a catalyst for the electroreduction of hydrogen peroxide to water at a potential between about xe2x88x920.1V and +0.4V versus the saturated calomel electrode (SCE) and causes the flow of an electrical current when the electrode is poised at a potential in this range. The magnitude of the current is then related to the concentration or the amount of amplified DNA.